Testicular germ cell tumors (TGCTs) are postulated to originate from carcinoma-in-situ (CIS) or intratubular germ cell neoplasia unclassified (ITGCNU) precursors. The mechanism(s) by which this pre-malignant precursor initiates and develops into both seminomas and nonseminomas is unknown. Various genetic studies have demonstrated that amplification of certain portion of chromosome 12p is consistently observed in the evolving germ cell tumor genome. The testis-specific protein Y-encoded (TSPY) gene is a tandemly repeated gene on the short arm of human Y chromosome. Most of its functional transcriptional units have been mapped within the critical region harboring the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Several studies have documented the expression of TSPY in the more common forms of TGCTs. To investigate the role of TSPY in the pathogenesis of TGCTs, we profiled the transcripts in CIS/ITGCNU and TGCT samples using GeneChip microarrays from Affymetrix. Analysis of microarray data demonstrated a correlation between TSPY expression and up-regulation of certain chromosome 12p.13 genes. These results support a role(s) for this Y chromosome gene in the pathogenesis of both gonadoblastoma and TGCTs.[unreadable] [unreadable] DNA methylation is one of the main epigenetic modifications during development. In cancer cells, its disruption is manifested in the regulatory regions (CpG islands) or histone proteins. Aberrant DNA methylation has been reported in testicular germ cell tumor (TGCT). The two sub-types of TGCT, namely, seminoma and non-seminoma share similar regional genomic disruptions. On the other hand, recent studies suggested different epigenotypes; CpG island methylation is virtually absent in seminomas while the methylation level in non-seminomas is similar to that of other solid tumors. Despite the random observation of aberrant methylation in TGCT, the global picture of epigenetic alteration in the TGCT genome remains unclear. To study epigenetic alterations in non-seminoma, we examined the complete genome methylation profile of several testicular cancer cell lines and non-seminoma tissues from patients by ChIP-on-chip using human tiling microarrays from Affymetrix. More than 40% of TGCT genome demonstrated genome-wide changes in methylation. Two gene clusters with pronounced differential methylation patterns were selected for confirmation of these observations. The first one is homeobox (Hoxa) gene cluster with extensive hypermethylation and the second one is pregnancy specific beta-1-glycoprotein (PSG) gene cluster with significant hypomethylation. Homeobox gene family is not only involved in segmentation during development but also in cell differentiation. Its epigenetic inactivation has been linked to the development of various human malignancies. PSGs are primarily expressed in placenta. However, over-expression of PSGs were observed in a wide variety of tumors and associated cell lines, particularly in colorectal, lung and brain tumors. We hypothesize the over-expression of PSGs in tumors may be due to the loss of transcription repression by hypomethylation and that PSGs could be used as a marker for TCGTs. We have collected a number of testicular tumor tissue samples, including archived frozen or paraffin samples of nonseminomas, seminomas, carcinoma in situ, as well as normal testis tissue samples for in-situ validation. The identification of novel epigenetic markers of TGCT, together with the development of user friendly and more sensitive assays, will improve the detection, treatment, and prognosis of this malignancy.